Immobilization of biomolecules on solid phases is widely used in numerous techniques such as e.g. in chromatography, in bio-sensors, in bio-reactors, e.g. for solid phase enzyme processing, chemical synthesis of peptides, oligonucleotides or other compounds and in so-called heterogeneous immunoassays.
In the heterogeneous immunoassays, antigens or antibodies can be covalently coupled to carriers such as cellulose, agarose or polyacrylamide. However, when the compounds are to be bound on a solid phase, usually polystyrene, polypropylene or polyvinylchloride test tubes or micro-titre-plates, physical adsorption of the compounds has been the normal coupling method in heteregeneous immunoassays (Engvall and Pearlmann, J. Immunol., 1972, p. 109-129).
Passive physical adsorption is, however, not irreversible (Lehtonen et al., J. Immunol. Methods, 1980, p. 34-61), which may affect the reproducibility of the immunoassays, especially when the antigen/antibody coated solid phase is stored in dry form. Also, solid phase adsorbed antigen may not be recognized by its corresponding antibody because of denaturation (reconformation) of the antigen tertiary structure (Kurki and Virtanen, J. Immunol. Methods, 1984, p. 67-122). The antigen may also exhibit new antigenic determinants when denatured, as in the case of DNA.
The ability of passive binding to plastic is furthermore restricted to a limited amount of molecules such as e.g. proteins, or single-stranded DNA. However, some proteins and nucleic acids as well as polysaccharides and smaller molecules cannot be adsorbed directly to some types of plastic.
Covalent binding, in contrast to simple physical binding, orientates all immobilized compounds in a defined way on the solid phase, thereby exposing defined areas e.g. antigens, antibodies or enzyme catalytic sites to the fluid phase eventually poured on the solid phase surface. Antigen epitopes or active sites on these compounds can in this way remain functional. Irreversible immobilization of molecules may furthermore have some advantages in relation to storage of solid phase immobilized compounds in their dry state, since passive physical binding partly denatures some proteins and nucleic acids.
For all these reasons it would be advantageous if it was possible to immobilize antigens, antibodies or other molecules covalently to the solid phase.
Covalent solid phase fixation of some types of compounds to polystyrene has been known for several years. Solid phase peptide and oligonucleotide syntheses are performed, for example, using modified polystyrene particles as the solid support. However, these modification methods frequently involve very hazardous chemicals and several time-consuming operation steps. An example of this is the preparation of Merrifield's peptide resin which has been widely used in peptide synthesis. Preparation of this resin involves the extremely carcinogenic reagent chloromethylmethyl ether and this and other organic solvents often result in a turbid surface because the plastic is slightly soluble in the reagents. This is inconvenient if a subsequent spectrophotometrical detection is desired.
Polystyrenes premodified with, for example, --OH, --SO.sub.3 H or --NH.sub.2 groups are available. When using these premodified polystyrenes a separate production of particles for each type of modified polystyrene is necessary.
A method for introducing amino groups on plastic surfaces for use in micro-titre-plates has been described (J. Virol. Methods 3, 1981, p. 155-165). This procedure, which requires the hazardous chemicals methanesulphonic acid, glacial acetic acid and fuming nitric acid, takes two days and needs a well functioning hood. Such conditions can hardly be used in large scale production due to environmental demands. Furthermore, these methods are generally time-consuming and it is difficult to obtain a uniform surface modification.
Chemical modification of polymer materials usually results in a heterogeneous mixture of products on the polymer surface since it is not possible to separate the solid phase bound main functional groups from the unwanted products of the reaction. This results more or less in a mixture of different active groups with different binding specificities to e.g. proteins.
Another method of chemically modifying or activating polymer surfaces by introducing functional groups such as OH, NH.sub.2, COOH, CHO, NCO or SCO is suggested in EP A patent application no. 155,252.
According to this application, a solid polymer surface is activated by radiation grafting of vinyl monomers having at least one functional group capable of binding to biologically active molecules. The polymer surface is grafted in solution in a chain transfer reducing solvent at a very low monomer concentration not exceeding 3% by weight, when the vinyl monomer is 1-mono-, or 1,1-disubstituted, in order to prevent homopolymerization and uncontrolled autocatalytic reactions. If the vinyl monomer is 1,2-substituted, higher concentrations of about 10% by weight may be used.
As applicable vinyl monomers are mentioned crotonic acid, acrylic acid, acrylic acid esters, acrylamide, bisacrylamide, methylol acrylamide, acrylated amines, acrylated epoxides, acrylated ethers, acrylated polyester, acrylated polyurethanes, acrylated acrylates, acrylated silicones, acrylated silanes, acrylated phosphates and acrylated titanates, acrolein, phenyl substituted styrene derivatives such as p-aminostyrene, tiglic acid, senecioic acid, angelic acid and cinnamic acid.
This process is difficult to control due to the inherent risk of excessive polymerization of the vinyl monomer. This is the most probable reason for the high binding capacity described. 5-7 .mu.g protein per well can probably not be immobilized as a monolayer in one single micro-titre-well. It is therefore essential that the vinyl monomer is present in a chain transfer reducing solvent, defined as a solvent which when irradiated forms radicals not able to initiate polymerization. As examples of suitable solvents are mentioned methanol, pyridine, water and mixtures of methanol and water. In practice, 1:1 methanol/water mixtures are used.
Thus the known process is based on the recognition that vinyl monomers, which are known to be polymerizable on polymer surfaces by irradiation graft polymerization or free radical graft polymerization, may be irradiation treated under conditions which prevent a polymerization and bound to the polymer surfaces as a thin graft layer close to a monomolecular layer leaving reactive groups capable of binding with biologically active molecules.
However, the known method suffer from a number of drawbacks. Firstly, the grafting process is very time-consuming. Even with .gamma.-radiation which is the only tested radiation source, the reaction time is 10-12 hours. .gamma.-radiation poses severe health physical requirements and there is a tendency of discolouring the plastic rendering it opaque and therefore inapplicable for optical measurements.
Further, the process requires an oxygen free atmosphere and a free radical initiator, for example, benzophenone.
The documentation provided in the examples does not convincingly show that immobilization of proteins has been accomplished due to covalent binding to the grafted polymer. Further, a standard coupling agent like glutaraldehyde or a carbodiimide reagent is used in most of the examples. These agents generally bring about a crosslinking of the protein molecules leading to an enhanced binding irrespective of the surface character.
Also, .gamma.-radiation is known to activate polymer surfaces and thereby improve their protein binding properties.
In conclusion it has not been unambiguously substantiated that the reported protein binding results are due to an activation of the polymer surface and not a result of the .gamma.-radiation and the use of coupling agents.
It has previously been described that bifunctional reagents containing arylazides can be bound to polymer surfaces (DE A 34 35 744). This was exemplified by adding protein A to a solid phase and subsequently adding the bifunctional compound: N-succinimidyl-6-(4-azido-2'-nitrophenyl-amino)-hexanoate. This results in binding of the arylazide derivative to the amino groups of protein A. This conjugate was subsequently exposed to light and a covalent binding to the polymer solid phase was postulated. However, there is no evidence of covalent binding between the polymer surface and protein A, since no data in the above-mentioned patent application showed the ability of protein A to bind to the polymer surface without addition of the arylazide compound.
Furthermore, during photolysis it is to be expected that degradation products of the azido group will more likely bind to the numerous nucleophilic groups present on protein A, thus forming aggregates of this protein. Furthermore, arylazides are known to react with nucleophiles as for example, water, thereby further reducing the likelihood for a possible reaction with the polymer surface.
Investigations have been carried out in order to biotinylate polystyrene photochemically using the commercial available reagent N-(4-azido-2-nitrophenyl)-N'-(3-biotinylamino-propyl)-N'-methyl-1,3-propan ediamine), (photobiotin, Sigma Cat. No. A 7667). This reagent contains an aryl azide group connected to a chemical linker similar to the one used according to EXAMPLE 1. The same optimizing experiments were performed as in this example, but no biotinylation of the polystyrene solid phase could be detected.